Circulating DNA has been identified in biological fluids (Lichtenstein et al., Ann NY Acad Sci., 945:239-49, 2001; Chan et al. Ann Clin Biochem, 40:122-30, 2003; Pathak et al. Clin Chem. 52:1833-42, 2006; Diehl et al. Nat Med. 14:985-90, 2008; Anker et al. Clin Chim Acta 313:143-6, 2001.) For example, in urine, two species are seen: a high-molecular-weight (HMW) urine DNA, greater than 1 kb, derived mostly from sloughed off cell debris from the urinary tract; and low-molecular-weight (LMW) urine DNA, approximately 150 to 250 base pairs (bp), derived primarily from apoptotic cells (Su et al. J Mol Diagn. 6:101-7, 2004.)
Hepatocellular carcinoma (HCC) is the most frequent cancer in certain parts of the world, and the fifth most cancer common worldwide. Increased incidence is associated with a spread in hepatitis C infections (El-Serag H B, Mason A C., N Engl J Med 340 (10):745-50, 1999). Surgical resection is often curative if the disease is localized and diagnosis occurs early (Klintmalm G B., Ann Surg 228 (4):479-90, 1998). However, extensive liver impairment associates with a poor prognosis, often due to late diagnosis. Among patients with underlying cirrhotic disease, a progressive increase in alpha-fetoprotein (AFP) and/or in alkaline phosphatase or a rapid deterioration of hepatic function may be the only clue to the presence of cancer.
Mutations in the p53 gene have been associated with approximately 50% of human cancers. In HCC, a G:C to T:A transversion at the codon 249 (249T) is a known “hotspot”. (Hsu et al. Nature, 350:377-8, 1991). Current methods of p53 detection such as restriction fragment length polymorphism and PCR-based assays followed by DNA sequencing are time consuming, labor intensive or insensitive. There remains a need for tests to screen for HCC that can detect the disease in early stages and be easily administered. The present invention provides methods for testing for p53 mutations and diseases associated with such types of mutations, e.g., HCC, using biological fluids such as urine. These uses as well as others will be apparent to those skilled in the art from the teachings herein.